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Transport properties of the human intestinal anion exchanger DRA (down-regulated in adenoma) in transfected HEK293 cells

  • Gastrointestinal Function
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Abstract

Electroneutral NaCl absorption in the intestine is mediated by parallel Na+/H+ and Cl−/HCO3− exchange. Mutations in the down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea but the transport characteristics of human DRA have not been studied in a heterologous human expression system. A N-terminal enhanced green fluorescence protein (EGFP)-tagged human DRA construct was therefore expressed stably in HEK293 cells. Cl−/HCO3− exchange was assessed by measuring intracellular pH and intracellular Cl− using fluorescent dyes. Expression of DRA resulted in the appearance of EGFP fluorescence and DRA immunoreactivity consistent with a location in the plasma membrane and possibly structures below the plasma membrane. DRA mediated electroneutral Cl−/HCO3− exchange but OH− was not transported and SO42−/HCO3− exchange was minimal. In the presence of 5% CO2/HCO3− the apparent affinity of DRA for Cl− in transfected HEK cells was 23–36 mM, which is lower than that reported for rabbit ileal brush border membrane vesicles and for oocytes injected with human DRA. DRA was inhibited by 4 mM DIDS (45±11%), by 50 µM tenidap (71±8%) and by 100 µM glibenclamide (59±22% inhibition of HCO3− transport and 79±3% inhibition of Cl− transport). The effects of DIDS and tenidap were not additive to those of glibenclamide.

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Fig. 1A–C
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Notes

  1. In pilot experiments tributyltin and nigericin were used to obtain a Stern-Volmer constant. Nevertheless to use this constant in individual experiments it would have been necessary to clamp [Cl−]i to 0 mM, again using the combination of tributyltin and nigericin. When this was tried the following experiments showed Cl− transport even in cells that had not shown Cl− transport previously indicating that tributylin/nigericin persisting in the perfusion system was contaminating the cells. The phenomenon disappeared only when the tubing was changed. Using a Stern-Vollmer constant for [Cl−]i measurements of 25 M−1 [20], our data indicate that resting [Cl−]i in HEK cells is 60–100 mM (see Figs. 4 and 6).

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Acknowledgements

Supported by grants La1066/2-1 and La 1066/2-3 from the Deutsche Forschungsgemeinschaft and by grant Fortune 548 of the University of Tübingen.

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  1. 1st Department of Medicine, University of Tübingen, 72076, Tübingen, Germany

    Georg Lamprecht, Susannah Baisch, Elena Schoenleber & Michael Gregor

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  1. Georg Lamprecht
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Correspondence to Georg Lamprecht.

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Lamprecht, G., Baisch, S., Schoenleber, E. et al. Transport properties of the human intestinal anion exchanger DRA (down-regulated in adenoma) in transfected HEK293 cells. Pflugers Arch - Eur J Physiol 449, 479–490 (2005). https://doi.org/10.1007/s00424-004-1342-x

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